Membrane fusion provides a conceptually simple mechanism for enveloped viruses to deliver their genomes into the cytoplasm of target cells. In contrast, the mechanisms used by nonenveloped viruses to penetrate cellular membranes and release their genomes into the cell remain poorly understood. This application proposes a program of structural, biochemical and biophysical studies of poliovirus cell entry intermediates as a simple model for understanding how nonenveloped viruses gain entry into the cell. The problem is framed in the context of several specific questions: 1) What is the nature of the virus-receptor interaction and how is receptor binding coupled to the induction of conformational changes? 2) What are the sequence of conformational changes that ultimately leads to cell entry and the release of the viral RNA? 3) What is the nature of the interaction of the virus with the cell membrane that leads to the internalization of the RNA? 4) How is the RNA released from the particle, across a vesicle membrane and into the cell? The central hypotheses governing this work are: that the machinery for this process with poliovirus will be directly applicable to closely related viruses (many of which including enterovirus 71 (EV71) and Coxsackievirus A16 (CAV16) are significant human pathogens): that some of features of the machinery will be conserved in more distantly related viruses, and that the nature of the machinery used by viruses will be relevant to understanding how nucleic acids and other large cargoes are transported across membranes in cells From a structural perspective, these questions cover over six orders of magnitude in scale from the atomic level (tenths of nanometers) to the cellular level (tens of microns). This range is well beyond the scope of any one structural method. The project seeks to develop a hybrid approach combining x-ray crystallography, cryoelectron microscopy, and cryoelectron tomography to develop a series of structural snapshots of cell entry intermediates of poliovirus; and to use these structures to address questions posed by, and pose questions for, supporting biochemical and genetic studies. The specific aims include structural studies of soluble cell entry intermediates in vitro t near-atomic resolution and of membrane-associated poliovirus cell entry intermediates in vitro at nanometer resolution. The aims also include the use of a simple receptor-decorated liposome model to identify the constituents of the channel that supports release of the genome across membranes and to characterize the kinetics and mechanism of RNA translocation at the single particle/single liposome level. The goal will be to define structural changes associated with cell entry and delivery of the genome into the cytoplasm at an unprecedented level of detail. The results are expected to be relevant to the design of novel antivirals and thermostable vaccines against poliovirus and related viruses including EV71 and CAV16.